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Article

Development and Validation of a Simple Analytical Method to Quantify Tocopherol Isoforms in Food Matrices by HPLC–UV–Vis

by
María Fernanda Arias-Santé
1,†,
Sussi López-Puebla
1,†,
Adriano Costa de Camargo
1,
José Luis Guil-Guerrero
2 and
Miguel Ángel Rincón-Cervera
1,2,*
1
Institute of Nutrition and Food Technology, University of Chile, Macul 7830490, Chile
2
Food Technology Division, University of Almería, 04120 Almería, Spain
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Appl. Sci. 2024, 14(19), 8750; https://doi.org/10.3390/app14198750
Submission received: 26 August 2024 / Revised: 25 September 2024 / Accepted: 26 September 2024 / Published: 27 September 2024
(This article belongs to the Special Issue New Insights into Bioactive Compounds)
Browse Figures
Versions Notes

Abstract

:
This study developed, validated, and tested a simple method for tocopherol analysis on five different food matrices (sunflower oil, mackerel fillets, almonds, spinach, and avocado pulp). Tocopherol extraction from foods was carried out by the Folch method and with n-hexane, and the identification and quantification of tocopherol isoforms (α, β, γ, and δ) was performed using normal-phase liquid chromatography with ultraviolet–visible detection (NP-HPLC–UV–Vis). The normal-phase column fully separated the four tocopherol isoforms in less than ten minutes. Linearity was shown to be excellent for the four isoforms in the assayed range (10–375 ppm, R2 > 0.99). Furthermore, the limits of detection (LOD) and quantification (LOQ) ranged from 0.32 to 0.63 ppm, and from 1.08 to 2.11 ppm, respectively. The intra-day and inter-day precision were assessed at different concentrations (10, 100, and 250 ppm) for each tocopherol isoform and they were within the range of acceptable values. Recovery rates were above 80% in most cases for all of the assayed food matrices, regardless of the extraction method (Folch solvents or n-hexane). α-Tocopherol was the main isoform found in all tested foods, and sunflower oil was the sample with the highest content, followed by almond, avocado pulp, mackerel fillet, and spinach. This method provides a convenient alternative for obtaining a complete profile of the four tocopherol isoforms in a variety of food matrices and for tracking the potential degradation kinetics of fortified foods during their processing and storage.

1. Introduction

Vitamin E is a group of lipid-soluble compounds known as tocochromanols or tocols. Tocols contain a chromanol ring as the primary structure, with a side chain at the C2 position and either methyl groups or hydrogen attached to the ring at the R1 or R2 locations, accounting for four tocopherol and four tocotrienol isoforms (α, β, γ, and δ) (Figure 1). Vitamin E is synthesized exclusively by photosynthetic organisms such as cyanobacteria, algae, and plants [1,2]. α-Tocopherol is the most active and abundant form of vitamin E in the human metabolism and it is considered an essential nutrient because it is not synthesized endogenously and must be supplied within the diet [3].
Vitamin E is known for its antioxidant capacity as a free radical scavenger, thus preventing lipid peroxidation [4]. Tocopherols also have a role in the prevention and treatment of cardiovascular diseases and certain types of cancer, as well as in improving immune function [5,6,7]. Cell signaling is one of the most important functions of α-tocopherol, allowing the integrity of polyunsaturated fatty acids in the cell membrane and suppressing hemolysis through stabilizing the red blood cell membrane [8].
Additionally, tocopherols help to increase the shelf life and stability of foods due to their antioxidant properties and interaction with polyunsaturated fatty acids in membrane lipids, protecting them against lipid peroxidation and scavenging reactive oxygen species (ROS) [2,9].
According to the Institute of Medicine of the US National Academies, the Dietary Reference Intakes (DRI) for α-tocopherol as the active form of vitamin E in humans are 5 mg (7.5 IU) for children between 1 and 3 years old, 6 mg (9.0 IU) for children between 4 and 10 years old, 9 mg (13.4 IU) for children between 11 and 14 years old, 12 mg (17.9 IU) for those older than 14 years, and 16 mg (23.9 IU) for breastfeeding women [10].
Vitamin E is found in several food groups. In general, nuts, seeds, vegetable oils, grains, and green leafy vegetables are considered good sources of vitamin E [2,9,11]. α-Tocopherol is found in high levels in green leafy vegetables, wheat germ and its oil, sunflower seeds and oil, rice bran, almonds, hazelnuts, grape seeds, and olive oil, while γ-tocopherol is found primarily in the seeds and oils of corn, rapeseed, canola, soybeans, and peanuts. Soybeans, sunflower seeds, and raspberries are also sources of δ-tocopherol [2,11].
Tocopherols are identified and quantified in foods by chromatographic methods, including gas chromatography, capillary electrochromatography, supercritical fluid chromatography, thin-layer chromatography, and normal- or reversed-phase high-performance liquid chromatography (NP/RP-HPLC) [12,13]. HPLC is a suitable and versatile tool for tocopherol analysis [11]. The combined use of different stationary phases and detection methods, such as light absorption (diode array or UV–Visible detectors), fluorescence, or mass spectrometry, allows a suitable separation of tocopherol isoforms with good reproducibility and precise identification and quantification [14,15,16,17].
This work aimed to validate a method for the identification and quantification of tocopherol isoforms (α, β, γ, and δ) in different foods (sunflower oil, mackerel fillet, almonds, avocado, and spinach) by HPLC–UV–Vis. Considering that the amount of tocotrienols in the selected food matrices is much lower than that of tocopherols [18,19,20,21,22], only the latter were analyzed in this study. Two lipid extraction methods (using either n-hexane or the Folch procedure with chloroform:methanol 2:1 v/v) were used for lipid extraction from fish, almonds, avocado, and spinach, and the results were compared in terms of vitamin E quantification. These foods were selected because they are considered to be good sources of vitamin E and to test for potential issues related to the matrix effect in tocopherol analysis in samples of different composition.

2. Materials and Methods

2.1. Chemicals

Individual analytical standards of α- (≥95% purity), β- (≥95% purity), γ- (≥95% purity), and δ-tocopherols (≥98% purity) were acquired from Cayman Chemical Co. (Ann Arbor, MI, USA). Unless otherwise stated, all other solvents and reagents were purchased from Merck (Darmstadt, Germany).

2.2. Samples

High oleic sunflower oil and samples of avocado, almonds, and spinach were acquired from local supermarkets in Santiago, Chile. Fillets of mackerel (Scomber japonicus) were provided by local fishermen in Coquimbo, Chile. Approximately 250 g of each sample (avocado pulp, almonds, spinach, and mackerel fillets) was crushed and homogenized in a high-power food processor (Ursus Trotter, model UT-METALLER605P 1.5 L, Santiago, Chile), frozen at −80 °C, and then lyophilized for 48 h (MFD-1050M model lyophilizer from MLAB, Chongqing, China) to remove water. The moisture content of each sample was recorded, and samples were then stored at −20 °C in sealed polyethylene bags under a nitrogen atmosphere until further processing.

2.3. Lipid Extraction

Lipid extraction from all of the assayed food matrices except sunflower oil was carried out by two procedures: the Folch method [23], and extraction with n-hexane [24]. For Folch extraction, 20 mL of chloroform:methanol (2:1 v/v) was added to 1 g of each lyophilized food and the mixture was magnetically stirred for 60 min under an inert nitrogen atmosphere and protected from light. After filtering to remove solid residues, the filtrate was collected and 4 mL of a magnesium chloride aqueous solution (9% w/v) was added. The two-phase system was allowed to completely separate in a separating funnel, and the lower organic phase was collected and filtered through anhydrous sodium sulfate. The solvent was then evaporated on a rotary evaporator at 40 °C (RV 3 V model from IKA, Staufen, Germany). The residue was collected, weighed, and stored at −20 °C protected from light and under an inert nitrogen atmosphere. In the case of n-hexane extraction, 10 mL of n-hexane was added to 1 g of each lyophilized food matrix and extracted for 30 min with magnetic stirring under an inert nitrogen atmosphere and protected from light. The mixture was then filtered and the filtrate was collected. The solid residue was extracted again with 10 mL n-hexane for 30 min. The solvent was removed in a rotary evaporator at 40 °C and the extracted lipids were collected, weighed, and stored at −20 °C protected from light and under an inert nitrogen atmosphere. All extractions were carried out in triplicate and results were reported as the mean value ± standard deviation.

2.4. Tocopherol Analysis

Analysis of tocopherol isoforms was performed on an Agilent 1260 HPLC with a UV–Vis detector (Agilent Technologies, Santa Clara, CA, USA). A Spherisorb Silica-80 column (250 × 4.6 mm, 5 μm particle size) from Waters (Milford, MA, USA) was used, and the mobile phase consisted of n-hexane:2-propanol (99:1 v/v) in isocratic mode at a flow rate of 1 mL/min. Samples (sunflower oil and the lipids extracted from fish, avocado, almonds, and spinach) were dissolved in n-hexane before being injected into the HPLC. The injection volume was 20 μL, and detection was performed at a wavelength of 292 nm. Tocopherol standards (α, β, γ, and δ) were injected separately to identify their retention time. Quantitative results of tocopherols in food matrices were reported in mg/100 g fresh weight (fw), considering the moisture content of all samples to convert data from dry weight (dw) to fw.

2.5. Method Validation

Standard calibration curves of each tocopherol isoform were built using seven known concentrations ranging from 10 to 375 μg/mL (ppm) in n-hexane. Peak identification was based on the retention time of each standard. The linearity range, limit of detection (LOD), limit of quantification (LOQ), recovery, and precision (intra- and inter-day) were evaluated. LOD and LOQ were estimated as 3 and 10 times the ratio of the standard deviation of the response to the slope of the calibration curve, respectively, according to the ICH Q2 (R1) validation guidelines. Intra-day precision was assessed at three different concentrations (10, 100, and 250 ppm) for each isoform (4 replicates on the same day), and inter-day precision was also tested at 10, 100, and 250 ppm for each isoform (4 replicates on 4 different days). The intra- and inter-day precision were reported as the percentage of the relative standard deviation (RSD%). The recovery percentage was determined by spiking each sample with two known concentrations of each tocopherol isoform before lipid extraction and chromatographic analysis according to the following formula:
R e c o v e r y   % = C o n c   f o u n d   i n   s p i k e d   s a m p l e − C o n c   f o u n d   i n   u n s p i k e d   s a m p l e S p i k e d   c o n c e n t r a t i o n × 100

2.6. Statistical Analysis

Experimental data were reported as the mean ± standard deviation (n = 3). GraphPad Prism 8 (La Jolla, CA, USA) statistical software was used. Differences between groups were assessed by the Student t-test and considered statistically significant at a p-value < 0.05.

3. Results and Discussion

3.1. Analysis of Tocopherol Isoforms by NP-HPLC–UV–Vis

The chromatographic method based on HPLC–UV–Vis with a normal-phase column provided a suitable separation and resolution of the four tocopherol isoforms with retention times of 5.04, 6.38, 6.73, and 8.70 min for α-, β-, γ-, and δ-tocopherols, respectively (Figure 2), showing a better performance than reversed-phase columns where β- and γ-tocopherols usually coelute and cannot be identified separately [25,26]. The assessment of linearity in the range of 10 to 375 μg/mL, LOD, and LOQ, as well as the intra-day and inter-day precision, are shown in Table 1.
All tocopherol isoforms exhibited an excellent linear response in the assayed range according to the R2 values. LOD and LOQ were in the ranges 0.32–0.63 and 1.08–2.11 ppm, respectively, for the four tocopherol isoforms, which are close to the values reported in previous works using HPLC-DAD as a chromatographic method for tocopherol analysis. Andrés et al. reported an LOD between 0.50 and 0.60 ppm and an LOQ between 1.5 and 2.0 ppm for different tocopherol isoforms (detection wavelength of 290 nm) [27], and Yuan et al. found an LOD between 0.20 and 0.35 ppm and an LOQ between 1.00 and 1.75 ppm (detection wavelength of 296 nm) [28]. Other studies have reported lower LOD and LOQ values for tocopherol isoforms when using fluorescence detection (FLD), such as Cruz and Casal (LOD of 0.006–0.007 ppm, LOQ of 0.019–0.023 ppm) [29], and Pokkanta et al. (LOD of 0.014–0.039 ppm and LOQ of 0.019–0.099 ppm) [30], due to the higher sensitivity of FLD compared to DAD or UV–Vis detectors. However, DAD and UV–Vis detectors are much more common in HPLC instrumentation than FLD due to their high versatility and applicability to the analysis of bioactive compounds such as sterols, carotenoids, organic acids, and vitamins in food matrices, in addition to their reliability and cost-effectiveness. Therefore, DAD and UV–Vis detectors are commonly used in many laboratories for tocopherol analysis [31].
The precision values were less than 15% for all of the assayed concentrations, which fits the acceptable limit for precision set by the Food and Drug Administration (FDA) [32]. This demonstrates that the method tested in this work is reliable for identifying and quantifying the four tocopherol isoforms.

3.2. Tocopherol Quantification in Food Matrices

In this study, vitamin E content was analyzed in five different food matrices: high oleic sunflower oil, spinach, almond, mackerel fillet, and avocado pulp (Table 2), which are usually considered rich sources of tocopherols according to the USDA Nutrient Database [33]. All samples, except sunflower oil, were subjected to lipid extraction either by the Folch method or with n-hexane before the chromatographic analysis, and the lipid extraction yields are depicted in Figure 3. Chromatograms of tocopherol analyses in all food matrices considering lipids extracted with the Folch method or with n-hexane are shown in Figures S1–S5.
It was found that α-tocopherol was the predominant isoform of vitamin E in all cases; sunflower oil was the sample with the highest amount of α-tocopherol (163.5 mg/100 g), and spinach showed the lowest amount (<0.20 mg/100 g). γ-Tocopherol was also found in lower proportions in all samples except sunflower oil. The presence of β- and δ-tocopherol was not detected in any matrix. The α-tocopherol content in sunflower oil used in this work was higher than that reported by other authors and higher than data available in food databases (~60–90 mg/100 g) [34,35,36]. It could be due to a fortification of the oil with α-tocopherol, which is sometimes carried out with unsaturated vegetable oils to prevent lipid oxidation and increase their shelf life [37].
Tocopherols are fat-soluble compounds and lipid extraction from the food matrix is usually carried out as a previous step to chromatographic analysis. The Folch method is considered the gold standard procedure for lipid extraction and involves using a mixture of chloroform and methanol [38]. Several studies have used Folch extraction to evaluate the tocopherol content in foods [39,40], but several other techniques have also been described to prepare samples before tocopherol analysis such as Soxhlet, alkaline hydrolysis, cold or hot mechanical extraction, saponification, and the use of other solvents such as hexane or methanol [19,41,42,43,44,45,46]. In this work, the analysis of tocopherol in different food matrices using Folch solvents or n-hexane for lipid extraction was compared, considering that using n-hexane offers a faster, cheaper, and simpler alternative and involves lower environmental and safety risks than Folch extraction [47]. Both methods were carried out at room temperature and have advantages over other commonly used methodologies for lipid extraction such as Soxhlet or those involving a saponification step, which are more time-consuming, require larger volumes of solvent, and are developed at higher temperatures, thus increasing the possibility of tocopherol degradation.
The lipid extraction yield was significantly higher using Folch extraction in mackerel fillet, almond, and spinach, but not in avocado pulp, where the values were close with both methods (Figure 3). However, despite the higher amount of extracted lipids from mackerel using the Folch method, the n-hexane extraction led to higher concentrations of α- and γ-tocopherols (1.33 and 0.61 mg/100 g, respectively) than Folch extraction (0.79 mg α-tocopherol/100 g). This fact might be due to potential interference in the chromatographic analysis of tocopherols caused by co-extracted compound/s from the fish matrix using the Folch method, which is a more efficient process to extract a wider variety of lipids including polar and non-polar compounds. Mackerel fillet showed values between 0.79 and 1.33 mg/100 g for α-tocopherol, which are within the range reported by other authors. Ribarova et al. found 1.59 mg/100 g of α-tocopherol in Bulgarian mackerel [40], Aminullah et al. found mackerel to have 1.30 mg/100 g of α-tocopherol [48], and Yerlikaya et al. reported 1.10 mg/100 g of α-tocopherol [46] when analyzed by FLD, UV–Vis detector, or DAD, respectively. Other studies have shown values around 0.4 mg/100 g of α-tocopherol evaluated by the same detection systems (UV–Vis or DAD) [49,50]. In the current work, γ-tocopherol was also found when lipids were extracted from fish with n-hexane (0.61 mg/100 g); however, other authors have not reported the content of this tocopherol isoform.
No significant differences in the concentration of α-tocopherol were found when using the Folch process (10.1 mg/100 g) and n-hexane (9.50 mg/100 g) for lipid extraction in almonds, despite the amount of lipids extracted with the Folch protocol being significantly higher than with n-hexane (52.5 and 44.7 g/100 g dw, respectively). However, a slight but significant increase in γ-tocopherol was noted using the Folch method (1.44 vs. 1.19 mg/100 g). Data regarding the tocopherol content in almonds are highly variable in the literature, considering that tocopherol concentration depends on the genotype, the environmental conditions, the irrigation practices, and the kernel maturation stage, among other factors. For example, values among 1.2 and 84.0 mg/100 g have been reported for α-tocopherol in almond kernels, which is the most abundant isoform of vitamin E in this food matrix [51,52]. Several studies have reported the amount of vitamin E in almond oil instead of in almond kernels, showing values in the range of 20–75 mg/100 g oil for α-tocopherol, and lower values for γ-tocopherol (0.2–7.5 mg/100 g oil) [11,41,42,51]. Whether concentrations of α- and γ-tocopherols are calculated on an oil basis in the current work (19–20 and 2.4–2.9 mg/100 g oil, respectively), such values are within the above-mentioned ranges.
The amounts of α-tocopherol found in spinach in the current work showed no significant differences between the lipid extraction methods, although the lipid extraction yield was slightly but significantly higher with the Folch method (0.63 g lipids/100 g dw) than using n-hexane (0.56 g lipids/100 g dw). γ-Tocopherol was only detected when carrying out the lipid extraction with n-hexane (0.09 mg/100 g fw). A broad signal was observed in the chromatogram of spinach lipids extracted by the Folch procedure at the retention time of γ-tocopherol (Figure S2), but the peak resolution was not considered good enough to proceed with the integration without the risk of overestimating the amount of γ-tocopherol in the sample. The Folch procedure involves the use of chloroform and methanol as extraction solvents with a wider range of polarity than when n-hexane is used, which probably resulted in the extraction of one or more compounds that overlapped the signal of γ-tocopherol, thus interfering with the proper identification of this tocopherol isoform. Tocopherol values reported in the literature in spinach samples are highly variable. For example, concentrations of α-tocopherol ranged between 1.6 and 3.9 mg/100 g fw in several studies [22,44,53,54,55], whereas much lower values (0.01–1.8 mg/100 g dw, converted into <0.001–0.12 mg/100 g fw considering an average moisture content of 93% in spinach) were found in other works [56,57]. Also, minor amounts of γ-tocopherol (0.06–0.30 mg/100 g fw) have been reported in spinach [22,44,55,57].
The avocado variety studied in this work (Chilean Hass) contained between 1.47 and 1.67 mg/100 g of α-tocopherol and between 0.04 and 0.46 mg/100 g of γ-tocopherol, with the α-tocopherol value being in the lower limit of the range reported in previous studies for avocado pulp (1.6–3.2 mg/100 g fw) [19,45,58]. Tocopherol amounts obtained using Folch extraction were significantly higher than with n-hexane and close to the values reported in the USDA Food Data Central Database (1.97 and 0.32 mg/100 g fw for α- and γ-tocopherol, respectively) [59].

3.3. Assessment of Recovery of Tocopherol in Food Matrices

All foods were spiked with a mix of the four tocopherol standards at two concentrations: one at the same concentration level of the main tocopherol isoform (α-tocopherol) found in unspiked analyzed samples and the other at twice that concentration. After calculating the concentration of each tocopherol isoform in each spiked sample using the calibration curves shown in Table 1, the recoveries were estimated in each case and the results are reported in Table 3.
The recoveries were in the ranges of 81.3–100.3% for α-tocopherol, 77.8–108.2% for β-tocopherol, 78.3–105.8% for γ-tocopherol, and 79.5–106.3% for δ-tocopherol. In all cases, recovery values were within the 70–120% range which is usually considered acceptable for most analytical method guidelines [32], although most recoveries were above 80%. This fact shows that both extraction methods are efficient in collecting tocopherols from all of the assayed food matrices.

4. Conclusions

The method developed and validated in this work provides a simple and efficient way to analyze α-, β-, γ-, and δ-tocopherol in different food matrices with high sensitivity and acceptable intra-day and inter-day precision and recovery using NP-HPLC with UV–Vis detection, which is one of the most common facilities available in laboratories conducting food analysis. Contrary to what is usually observed when using reversed-phase chromatographic columns, the use of a normal-phase column with an isocratic program allowed the complete separation of the four tocopherol isoforms in less than 10 min. The common problem of baseline drifting caused by differences in the UV absorbance of mobile phase solvents when working with a gradient method was avoided in this study by using an isocratic program throughout the analysis. In addition, two lipid extraction methods were compared to verify their suitability in terms of tocopherol quantification in different food matrices. Significantly higher amounts of α- and γ-tocopherol were found in the avocado pulp when using the Folch method for lipid extraction instead of n-hexane. Conversely, in almonds, only the amount of γ-tocopherol was found to be significantly higher using Folch extraction compared to n-hexane. On the other hand, significantly higher levels of α- and γ-tocopherol were found in mackerel fillets when n-hexane was used to extract the lipid fraction instead of the Folch method. Neither β- nor δ-isoforms were found in any of the analyzed matrices.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/app14198750/s1, Figure S1: HPLC-UV-vis chromatograms of unspiked (A) and spiked (B) high-oleic sunflower oil with the four tocopherol isoforms (α, β, γ and δ) measured at 292 nm; Figure S2: HPLC-UV-vis chromatograms of the lipid fraction extracted with the Folch method (A) or n-hexane (B) of unspiked spinach, and the lipid fraction of spiked spinach with the four tocopherol isoforms (α, β, γ and δ) (C) measured at 292 nm; Figure S3: HPLC-UV-vis chromatograms of the lipid fraction extracted with the Folch method (A) or n-hexane (B) of unspiked avocado pulp, and the lipid fraction of spiked avocado pulp with the four tocopherol isoforms (α, β, γ and δ) (C) measured at 292 nm; Figure S4: HPLC-UV-vis chromatograms of the lipid fraction extracted with the Folch method (A) or n-hexane (B) of unspiked almond, and the lipid fraction of spiked almond with the four tocopherol isoforms (α, β, γ and δ) (C) measured at 292 nm; Figure S5: HPLC-UV-vis chromatograms of the lipid fraction extracted with the Folch method (A) or n-hexane (B) of unspiked mackerel fillet, and the lipid fraction of spiked mackerel fillet with the four tocopherol isoforms (α, β, γ and δ) (C) measured at 292 nm.

Author Contributions

Conceptualization, M.Á.R.-C.; Methodology, A.C.d.C. and M.Á.R.-C.; Validation, M.F.A.-S. and S.L.-P.; Formal analysis, M.F.A.-S. and S.L.-P.; Investigation, M.F.A.-S. and S.L.-P.; Resources, M.Á.R.-C.; Writing—original draft preparation, M.F.A.-S., S.L.-P., J.L.G.-G. and M.Á.R.-C.; Writing—review and editing, M.F.A.-S., S.L.-P., A.C.d.C., J.L.G.-G. and M.Á.R.-C.; Supervision, M.Á.R.-C.; Project administration, M.Á.R.-C.; Funding acquisition, M.Á.R.-C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Fund for Scientific and Technological Development (FONDECYT), Grant Number 1220532.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Materials, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 08750 g001
Figure 1. Chemical structures of the vitamin E isoforms.
Figure 1. Chemical structures of the vitamin E isoforms.
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Applsci 14 08750 g002
Figure 2. HPLC–UV–Vis chromatogram of standards of the four tocopherol isoforms (600 ppm each) measured at λ = 292 nm.
Figure 2. HPLC–UV–Vis chromatogram of standards of the four tocopherol isoforms (600 ppm each) measured at λ = 292 nm.
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Figure 3. Lipid extraction yields (g/100 g dw) of mackerel fillets, almonds, spinach, and avocado pulp using n-hexane or the Folch method. Within each sample, * means a significant difference between the extraction yields found by the two assayed methods (Student t-test, p < 0.05).
Figure 3. Lipid extraction yields (g/100 g dw) of mackerel fillets, almonds, spinach, and avocado pulp using n-hexane or the Folch method. Within each sample, * means a significant difference between the extraction yields found by the two assayed methods (Student t-test, p < 0.05).
Applsci 14 08750 g003
Table 1. Method validation parameters for the analysis of tocopherol isoforms.
Table 1. Method validation parameters for the analysis of tocopherol isoforms.
CompoundRegression
Equation 		Linearity Coefficient (R2)LOD
(ppm) 		LOQ
(ppm)		Intra-Day
Precision (RSD%)		Inter-Day
Precision (RSD%)
α-tocopheroly = 7.549x − 61.1830.99880.381.281.79 (at 10 ppm)
1.50 (at 100 ppm)
1.87 (at 250 ppm)		3.06 (at 10 ppm)
1.81 (at 100 ppm)
1.54 (at 250 ppm)
β-tocopheroly = 4.206x − 34.3240.99850.321.082.84 (at 10 ppm)
2.59 (at 100 ppm)
1.66 (at 250 ppm)		4.43 (at 10 ppm)
2.51 (at 100 ppm)
4.32 (at 250 ppm)
γ-tocopheroly = 8.144x − 75.8550.99840.622.062.56 (at 10 ppm)
2.82 (at 100 ppm)
1.44 (at 250 ppm)		7.80 (at 10 ppm)
7.77 (at 100 ppm)
5.05 (at 250 ppm)
δ-tocopheroly = 6.361x − 56.7390.99880.632.113.63 (at 10 ppm)
1.39 (at 100 ppm)
2.15 (at 250 ppm)		7.51(at 10 ppm)
7.40 (at 100 ppm)
6.05 (at 250 ppm)
Table 2. Tocopherol quantification (mg/100 g fw) in five food matrices. Different superscript letters within the same food type for each tocopherol isoform indicate significant differences between values (Student t-test, p < 0.05).
Table 2. Tocopherol quantification (mg/100 g fw) in five food matrices. Different superscript letters within the same food type for each tocopherol isoform indicate significant differences between values (Student t-test, p < 0.05).
Food MatrixExtraction Methodα-Tβ-Tγ-Tδ-T
High Oleic
Sunflower Oil 		-163.5 ± 2.3n.d.n.d.n.d.
Mackerel Fillet Folch
n-hexane		0.79 ± 0.05 a
1.33 ± 0.05 b		n.d.
n.d.		n.d.
0.61 ± 0.02		n.d.
n.d.
AlmondFolch
n-hexane		10.1 ± 0.5
9.50 ± 0.15		n.d.
n.d.		1.44 ± 0.04 a
1.19 ± 0.05 b		n.d.
n.d.
SpinachFolch
n-hexane		0.16 ± 0.01
0.17 ± 0.01		n.d.
n.d.		n.d.
0.09 ± 0.01		n.d.
n.d.
AvocadoFolch
n-hexane		1.67 ± 0.02 a
1.47 ± 0.01 b		n.d.
n.d.		0.46 ± 0.01 a
0.04 ± 0.01 b		n.d.
n.d.
Table 3. Recovery values (%) of each tocopherol isoform in the five assayed foods after fortification with α-, β-, γ-, and δ-tocopherols at two concentration levels.
Table 3. Recovery values (%) of each tocopherol isoform in the five assayed foods after fortification with α-, β-, γ-, and δ-tocopherols at two concentration levels.
Food MatrixExtraction MethodFortification Level (mg/100 g fw) Recovery (%)
α-Tβ-Tγ-Tδ-T
High Oleic
Sunflower Oil		-160100.3 ± 1.2108.2 ± 2.6105.5 ± 2.2105.6 ± 2.4
-320100.3 ± 1.4106.2 ± 2.5105.8 ± 2.3106.3 ± 1.7
Mackerel
fillet		Folch0.8087.8 ± 2.988.6 ± 4.1104.4 ± 4.398.0 ± 3.9
1.6086.8 ± 3.690.7 ± 4.786.0 ± 3.589.1 ± 1.9
n-hexane1.3093.3 ± 3.285.9 ± 6.995.4 ± 3.682.8 ± 6.5
2.6094.2 ± 1.982.9 ± 3.490.5 ± 1.879.5 ± 3.3
Almond Folch10.081.9 ± 2.677.8 ± 3.482.9 ± 0.980.3 ± 4.5
20.085.8 ± 0.783.9 ± 2.281.3 ± 2.588.8 ± 1.2
n-hexane10.089.1 ± 2.078.0 ± 2.184.5 ± 2.486.2 ± 0.9
20.092.6 ± 1.387.4 ± 1.686.2 ± 2.291.8 ± 0.6
SpinachFolch0.1681.8 ± 2.489.2 ± 2.386.6 ± 5.788.4 ± 1.6
0.3287.0 ± 1.085.1 ± 1.487.7 ± 3.486.2 ± 2.9
n-hexane0.1685.7 ± 1.989.2 ± 3.884.9 ± 2.480.6 ± 3.3
0.3287.0 ± 1.085.1 ± 1.487.6 ± 2.686.2 ± 2.9
Avocado Folch1.5082.4 ± 0.785.4 ± 3.178.3 ± 1.682.3 ± 2.5
3.0081.3 ± 0.584.1 ± 1.580.4 ± 0.488.7 ± 3.4
n-hexane1.5089.2 ± 2.189.0 ± 3.283.3 ± 4.086.2 ± 2.9
3.0089.3 ± 0.579.0 ± 4.086.8 ± 1.790.0 ± 2.9
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Arias-Santé, M.F.; López-Puebla, S.; de Camargo, A.C.; Guil-Guerrero, J.L.; Rincón-Cervera, M.Á. Development and Validation of a Simple Analytical Method to Quantify Tocopherol Isoforms in Food Matrices by HPLC–UV–Vis. Appl. Sci. 2024, 14, 8750. https://doi.org/10.3390/app14198750

AMA Style

Arias-Santé MF, López-Puebla S, de Camargo AC, Guil-Guerrero JL, Rincón-Cervera MÁ. Development and Validation of a Simple Analytical Method to Quantify Tocopherol Isoforms in Food Matrices by HPLC–UV–Vis. Applied Sciences. 2024; 14(19):8750. https://doi.org/10.3390/app14198750

Chicago/Turabian Style

Arias-Santé, María Fernanda, Sussi López-Puebla, Adriano Costa de Camargo, José Luis Guil-Guerrero, and Miguel Ángel Rincón-Cervera. 2024. "Development and Validation of a Simple Analytical Method to Quantify Tocopherol Isoforms in Food Matrices by HPLC–UV–Vis" Applied Sciences 14, no. 19: 8750. https://doi.org/10.3390/app14198750

APA Style

Arias-Santé, M. F., López-Puebla, S., de Camargo, A. C., Guil-Guerrero, J. L., & Rincón-Cervera, M. Á. (2024). Development and Validation of a Simple Analytical Method to Quantify Tocopherol Isoforms in Food Matrices by HPLC–UV–Vis. Applied Sciences, 14(19), 8750. https://doi.org/10.3390/app14198750

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